Abstract

Purpose: The process of optimization and fabrication of nanoparticle synthesis for preclinical studies can be challenging and time consuming. Traditional small scale laboratory synthesis techniques suffer from batch to batch variability. Additionally, the parameters used in the original formulation must be re-optimized due to differences in fabrication techniques for clinical production. Several low flow microfluidic synthesis processes have been reported in recent years for developing nanoparticles that are a hybrid between polymeric nanoparticles and liposomes. However, use of high flow microfluidic synthetic techniques has not been described for this type of nanoparticle system, which we will term as nanolipomer. We hypothesize that it is possible to manufacture nanolipomers in large batches using a high flow microfluidic synthesis method and these nanoplipomers will maintain optimal physico-chemical and functional parameters.

Methods: Nanolipomers were synthesized through a microfluidic process utilizing the Nanoassembler platform. Nanolipomer size and zeta potential were measured through dynamic light scattering techniques. Time resolved lifetime and anisotropy experiments were performed to verify drug loading. MTT assay was performed on C4-2B prostate cancer cells to assess cell viability after treatment with nanolipomers. Nude mice were intravenously injected with nanolipomers to determine in vivo biocompatibility.

Results: The optimal total flow rate for synthesis of these nanolipomers was found to be 12 ml/min and flow rate ratio 1:1 (organic phase: aqueous phase). The PLGA polymer concentration of 10 mg/ml and a DSPE-PEG lipid concentration of 10% w/v provided optimal size, PDI and stability. Drug loading and encapsulation of a representative hydrophobic small molecule drug, curcumin, was optimized and found that high encapsulation efficiency of 58.8% and drug loading of 4.4% was achieved at 7.5% w/w initial concentration of curcumin/ PLGA polymer. The final size and polydispersity index of the optimized nanolipomer was 102.11 nm and 0.126, respectively. Functional assessment of uptake of the nanolipomers in C4-2B prostate cancer cells showed uptake at 1 hour and increased uptake at 24 hours. The nanolipomer was more effective in the cell viability assay compared to free drug. Finally, assessment of in vivo retention in mice of these nanolipomers revealed retention for up to 2 hours and were completely cleared at 24 hours.

Conclusions: In this study, we have demonstrated that a nanolipomer formulation can be successfully synthesized and easily scaled up through a high flow microfluidic system with optimal characteristics. The process of developing nanolipomers using this methodology is significant as the same optimized parameters used for small batches could be translated into manufacturing large scale batches for clinical trials through parallel flow systems.

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Cancer

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Scale up of nanolipomer microfluidic production for potential clinical trials

Purpose: The process of optimization and fabrication of nanoparticle synthesis for preclinical studies can be challenging and time consuming. Traditional small scale laboratory synthesis techniques suffer from batch to batch variability. Additionally, the parameters used in the original formulation must be re-optimized due to differences in fabrication techniques for clinical production. Several low flow microfluidic synthesis processes have been reported in recent years for developing nanoparticles that are a hybrid between polymeric nanoparticles and liposomes. However, use of high flow microfluidic synthetic techniques has not been described for this type of nanoparticle system, which we will term as nanolipomer. We hypothesize that it is possible to manufacture nanolipomers in large batches using a high flow microfluidic synthesis method and these nanoplipomers will maintain optimal physico-chemical and functional parameters.

Methods: Nanolipomers were synthesized through a microfluidic process utilizing the Nanoassembler platform. Nanolipomer size and zeta potential were measured through dynamic light scattering techniques. Time resolved lifetime and anisotropy experiments were performed to verify drug loading. MTT assay was performed on C4-2B prostate cancer cells to assess cell viability after treatment with nanolipomers. Nude mice were intravenously injected with nanolipomers to determine in vivo biocompatibility.

Results: The optimal total flow rate for synthesis of these nanolipomers was found to be 12 ml/min and flow rate ratio 1:1 (organic phase: aqueous phase). The PLGA polymer concentration of 10 mg/ml and a DSPE-PEG lipid concentration of 10% w/v provided optimal size, PDI and stability. Drug loading and encapsulation of a representative hydrophobic small molecule drug, curcumin, was optimized and found that high encapsulation efficiency of 58.8% and drug loading of 4.4% was achieved at 7.5% w/w initial concentration of curcumin/ PLGA polymer. The final size and polydispersity index of the optimized nanolipomer was 102.11 nm and 0.126, respectively. Functional assessment of uptake of the nanolipomers in C4-2B prostate cancer cells showed uptake at 1 hour and increased uptake at 24 hours. The nanolipomer was more effective in the cell viability assay compared to free drug. Finally, assessment of in vivo retention in mice of these nanolipomers revealed retention for up to 2 hours and were completely cleared at 24 hours.

Conclusions: In this study, we have demonstrated that a nanolipomer formulation can be successfully synthesized and easily scaled up through a high flow microfluidic system with optimal characteristics. The process of developing nanolipomers using this methodology is significant as the same optimized parameters used for small batches could be translated into manufacturing large scale batches for clinical trials through parallel flow systems.